Activation and signaling mechanism revealed by GPR119-Gs complex structures

Agonists selectively targeting cannabinoid receptor-like G-protein-coupled receptor (GPCR) GPR119 hold promise for treating metabolic disorders while avoiding unwanted side effects. Here we present the cryo-electron microscopy (cryo-EM) structures of the human GPR119-Gs signaling complexes bound to AR231453 and MBX-2982, two representative agonists reported for GPR119. The structures reveal a one-amino acid shift of the conserved proline residue of TM5 that forms an outward bulge, opening up a hydrophobic cavity between TM4 and TM5 at the middle of the membrane for its endogenous ligands-monounsaturated lipid metabolites. In addition, we observed a salt bridge between ICL1 of GPR119 and Gβs. Disruption of the salt bridge eliminates the cAMP production of GPR119, indicating an important role of Gβs in GPR119-mediated signaling. Our structures, together with mutagenesis studies, illustrate the conserved binding mode of the chemically different agonists, and provide insights into the conformational changes in receptor activation and G protein coupling.

and other places.
Reviewer #4: Remarks to the Author: Qian et al. provide structural context for the active state of human GPR119 in the presence of two synthetic agonists and the receptor's cognate G protein. The authors present two high quality cryoelectron microscopy structures of the receptor and identify structural features that have not been reported for other class A G protein-coupled receptors, including an outward bulge in transmembrane helix 5 and a salt-bridge between intracellular loop 1 and the G subunit. Other protein-ligand interactions that could be relevant for GPR119 activation and signaling are identified from the structures and then experimentally verified through multiple point mutations. The authors also explore the binding mode of different agonists and their derivatives using a docking study and perform molecular dynamics simulations where they compare the dynamics of the ligand-free vs. the agonistbound receptor. Overall, I believe that the study is thorough and that the reported findings will be of great interest for the G protein-coupled receptor community. However, the present manuscript would benefit from some revisions, particularly pertaining to the following points: Observations related to the computational work 1. Page 21, line 14-16: It needs to be clarified what subset of atoms was included in the RMSD calculations that are reported in Figure 12a. The loops of GPCRs tend to be quite flexible and it is customary to exclude them when checking the stability of the system. Most groups report the RMSD of the C s in the transmembrane domain. It is not clear from the text or the figure if this is what the authors did or not? 2. Page 32, line 24: The authors should provide a more complete description of how the initial structure of GPR119 used for refinement was generated. What templates did they use? What were their percentages of identity vs. GPR119? How many models did they generate with MODELLER and how were they ranked (e.g., based on their DOPE score)? 3. Page 34, line 5: Can the authors elaborate on the need to use rigid docking for OEA, but not for the other derivatives that were docked? 4. Page 34, line 5: Please provide the docking scores obtained from the docking studies. 5. Page 34, line 20: Do the authors mean "adapted" or "adopted"? Were there penalties higher than 10 in the parameters generated from CGenFF? If so, were the parameters optimized as suggested by Vanommeslaeghe et al. (2012) in their J. Chem. Inf Model 52(12), 3144-54 paper? 6. SI Page 12, line 4: How were the average/representative structures presented in Supplementary  Figures 11b, 11d, 12c, 12e and 12f generated? Since it is not really meaningful to average coordinates, usually one would cluster the conformations sampled in the simulations and use the medoid from the most populated cluster as a representative structure.

Other observations
A. Missing references 1. Page 7, line 5: The reference to the Ballesteros-Weinstein paper needs to be added.
B. Data presentation: 1. Page 6, line 12-14: Figure 1 does not really show the two ligands overlapping, but in different panels. And would the fact that they overlap demonstrate that the activation mechanism is conserved? I think that this sentence needs to be reworded to reflect what it is actually in the figure. 2. Figure 2e and f: The different cavities of the ligand binding pocket need to be more clearly defined, because their limits are not clear just based on the arrows shown in the panels. Perhaps it would be helpful if they were indicated with boxes instead? 3. Figure 3a-i: Adding BW-numberings to the residues would be helpful for the reader and it would also match the notation used in the bar plots. 4. Figure 4: Since this figure includes mutations in both the receptor and the G protein, I would suggest adding a prefix so that they are easier to identify (e.g., R_ for receptor residues, G _ for G residues and so on). 5. Supplementary Table 1: The value of the rotamer outliers for GPR119-MBX-Gs-Nb35 needs to be centered.

Response to reviewers' specific comments:
Reviewer #1 (Remarks to the Author): In my initial review (for NSMB), I requested that the authors try and get the unliganded or apo state structure for GPR119. I understand that it has been technically challenging to get it and I have nothing more to ask for experiments. I support publication of the paper in its current form, with some language formatting. The quality of the structures seems very good, and overall it adds to our knowledge of this orphan GPCR.
We appreciate the reviewer's positive evaluation of our work and its significance. We have addressed all of his/her comments with significant changes to the manuscript.

All have been corrected as suggested.
Reviewer #2 (Remarks to the Author): In response to the questions raised, the authors basically solved my doubts. The description of the content of manuscript has been modified and the format of figure has been optimized, which make the text more logical and clear. However, some words in the manuscript are not precise. For example, Page 6, line 5, "for all amino acids": it is better to use "for most amino acids".

This sentence has been corrected as suggested.
Page 8, line 21, "with the deepest depth… thus far": it will be more appropriate to use "deeper…than" for these descriptions.

This sentence has been corrected as suggested.
Other similar parts should be checked if I didn't point out.

Reviewer #3 (Remarks to the Author):
Reply from Reviewer 3 The manuscript has been improved with respect to a biological context of GPR119. However, the authors may consider to update the view here with the more recent literature, for instance doi.org/10.1186/s11658-021-00276-7.

The recent literature has been updated and cited.
The author could also improve the manuscript by providing overview of the two selected compounds and their path as putative lead compounds for GPR119 We would like to clarify that we have provided overview of the two selected compounds in the introduction part, and have discussed SAR for many compounds (supplemental Supplementary Fig. 6). These would provide a path as putative lead compounds for GPR119.
Moreover, the authors may want to correct a few language/typographic mistakes, for instance: -page 5: line 5, line 8 and 9. -page 11: line 2 -page 15: line 16 and other places.

All have been corrected as suggested.
Reviewer #4 (Remarks to the Author): Qian et al. provide structural context for the active state of human GPR119 in the presence of two synthetic agonists and the receptor's cognate G protein. The authors present two high quality cryoelectron microscopy structures of the receptor and identify structural features that have not been reported for other class A G protein-coupled receptors, including an outward bulge in transmembrane helix 5 and a salt-bridge between intracellular loop 1 and the G훽 subunit. Other protein-ligand interactions that could be relevant for GPR119 activation and signaling are identified from the structures and then experimentally verified through multiple point mutations. The authors also explore the binding mode of different agonists and their derivatives using a docking study and perform molecular dynamics simulations where they compare the dynamics of the ligand-free vs. the agonist-bound receptor. Overall, I believe that the study is thorough and that the reported findings will be of great interest for the G protein-coupled receptor community. However, the present manuscript would benefit from some revisions, particularly pertaining to the following points: We appreciate the reviewer's positive evaluation of our work and its significance. We have addressed all of his/her comments with significant changes to the manuscript.  Figure R1). As expected, the current RMSD values are much smaller than previous data, no more than 2.5 Å. This is consistent with our conclusion that GPR119 reached stability during the simulation. Thus, we've replaced Figure 11a with the new results below. 2. Page 32, line 24: The authors should provide a more complete description of how the initial structure of GPR119 used for refinement was generated. What templates did they use? What were their percentages of identity vs. GPR119? How many models did they generate with MODELLER and how were they ranked (e.g., based on their DOPE score)?
In the docking studies, we have applied two strategies, rigid docking and flexible docking, for all ligands. As shown in Fig. 3g, 3h and 3i, OEA is long and thin, which means it has a smaller volume and could exhibits more conformational flexibility compared to AR231453 or MBX-2982. Therefore, rigid docking is already enough to obtain good, comparable binding poses for OEA. To better compare OEA's binding mode with AR231453 or MBX-2982, we chose to use the rigid docking results, for surrounding residues display identical conformations in these complexes. For the other derivatives, most of them has additional moieties compared with AR231453 or MBX-2982 ( Supplementary Fig. 6). Their larger molecular volume causes steric hindrance in the binding pocket and only flexible docking could produce reasonable, comparable binding poses. Though very individual derivatives with smaller volume could also obtain binding conformations under rigid docking, to maintain the consistency in comparison, only flexible docking results were shown for each derivative in Supplementary Figure 7. According to the above description, we've included corresponding explanations in the method section. 3. Page 34, line 5: Can the authors elaborate on the need to use rigid docking for OEA, but not for the other derivatives that were docked?
In the docking studies, we have applied two strategies, rigid docking and flexible docking, for all ligands. As shown in Fig. 3g, 3h and 3i, OEA is long and thin, which means it has a smaller volume and could exhibit more conformational flexibility compared to AR231453 or MBX-2982. Therefore, rigid docking is already enough to obtain good, comparable binding poses for OEA. To better compare OEA's binding mode with AR231453 or MBX-2982, we chose to use the rigid docking results, for surrounding residues display identical conformations in these complexes. For the other derivatives, most of them has additional moieties compared with AR231453 or MBX-2982 (Supplementary Fig. 6)

Docking scores generated from the docking studies are listed below. We didn't provide this table in the manuscript because there is no correlation between docking scores and EC50 of ligands, which may make the readers confusing. This is understandable because the scoring of the docking conformation by Autodock Vina is based on the semi-empirical free energy scoring function, which has some limitations in different situations, and higher scores only indicate better binding interactions
between the compounds and receptors in specific computational models but have no direct correlation with experimental activity. Therefore, the most reliable (or representative) binding poses shown in figures were selected according to both the docking scores and their frequency of occurrence in all obtained conformations for each ligand. For example, if the top-scoring pose only appears once, it will not be considered as a reliable binding pose. In the tables below, we listed the docking score of the selected pose, which is usually not the one with the best score, as well as the range of the docking score for all conformations. It's obvious that docking scores have no relevance to their efficacy from any perspective. To avoid making readers confusing, we'd better not present these scores in the manuscript.  Supplementary Fig 6b  3.4 -7.0 -7.8 -5.8 Supplementary Fig 6c  110 -7.5 -7.9 -6.0 Supplementary Fig 6d  7 -7.7 -8.2 -6.1 Supplementary Fig 6e  260 -7.3 -8.0 -6.0 Supplementary Fig 6f  10 -7.4 -7.7 -5.4 Supplementary Fig 6g  1.5 -6.8 -7.7 -6.0 Supplementary Fig 6i  182 -9.4 -10.0 -8.1 Supplementary Fig 6j  49 -10.0 -11.3 -3.6 Supplementary Fig 6k  100 -10.6 -10.6 -8.2 a EC50 values of derivatives were obtained from previous studies. We thank the reviewer for the insightful comment. We've changed "adapted" to "adopted" in the corresponding text. With regard to the ligand parameters, there are penalties higher than 10, but unfortunately, we didn't perform optimization at that time. The parameters assigned by CGenFF are listed in detail in the Ligand Parameters section in the Supporting material. Your suggestion is undoubtedly the best in theory, but it needs us to rerun the simulation with optimized parameters, which is usually very expensive in terms of computational resources and time costs. Due to the huge amount of simulation time and computational resources required, we couldn't provide the updated data in the current response. Furthermore, we invite the reviewer to consider our following discussion before doing it: (1) The purpose of MD simulation in this work is to make more adequate structural comparison between agonist-bound and the predicted ligand-free GPR119 besides static structures. Especially for the predicted AlphaFold structure, MD simulation could optimize its conformation. By comparing conformations of the transmembrane domain as well as ligand binding pocket both from simulation and from static structure, we would be able to draw more convincing conclusions about structural features related to agonist bound. Given that the comparison among static structures already exists ( Supplementary Fig. 10), simulation data only plays a supporting role. 6. SI Page 12, line 4: How were the average/representative structures presented in Supplementary  Figures 11b, 11d, 12c, 12e and 12f generated? Since it is not really meaningful to average coordinates, usually one would cluster the conformations sampled in the simulations and use the medoid from the most populated cluster as a representative structure.
We thank the reviewer for his/her insightful comment. In Supplementary  Figure 11b Fig. 12d). In this case, snapshots in the dominant conformation presenting similar RMSD values could be considered as representative structures, which actually works something like conformation clustering. Based on the above reasons, the snapshot at 900 ns was suitable to be a typical structure for each simulation, so we finally extracted them as representative structures and illustrated their dynamic features in Supplementary Figure 12b, 12c, 12e and 12f. Following the reviewer's suggestions, we conducted conformation clustering to show more precise results in modified Supplementary Figure 11 and 12. Protein conformations were clustered based on the RMSD values of transmembrane domain (shown in Supplementary Fig.  11a) using the last 200 ns snapshots in each simulation. The middle structure from the largest cluster was chosen as the representative structure and used for the superimposition in Supplementary Figure 11b and 11d. To present typical ligand binding poses and their surrounding residues, we clustered ligand conformations sampled in the last 200 ns using the ligand RMSD (shown in Supplementary Fig. 12a and 12d). Typical structures were determined in the same way. They were used to illustrate ligand binding conformations in Supplementary Figure 12c and 12f and to present pocket residues in Supplementary Figure 12b    The reference has been added.
B. Data presentation: 1. Page 6, line 12-14: Figure 1 does not really show the two ligands overlapping, but in different panels. And would the fact that they overlap demonstrate that the activation mechanism is conserved? I think that this sentence needs to be reworded to reflect what it is actually in the figure.
We have reworded this sentence to reflect what it is actually in the figure.
2. Figure 2e and f: The different cavities of the ligand binding pocket need to be more clearly defined, because their limits are not clear just based on the arrows shown in the panels. Perhaps it would be helpful if they were indicated with boxes instead?
We have changed the arrows with circles to clearly define the different cavities. 3. Figure 3a-i: Adding BW-numberings to the residues would be helpful for the reader and it